The present invention relates to cloned DNA encoding the Tsp45I restriction endonuclease (Tsp45I) as well as the Tsp45I modification methylase (M. Tsp45I), and the production of recombinant Tsp45I.
Many species of bacteria contain small circular extrachromosomal genetic elements, known as plasmids. Plasmids have been found in a number of bacteria which live in extreme environments, including the thermophiles, which live at high (&gt;55.degree. C.) temperatures (Munster et al., Appl. Environ. Microbiol. 50:1325-1327 (1985); Kristjansson and Stetter, in `Thermophilic Bacteria`, Kristjansson, ed., p. 1-18 (1992)). However, most thermophile plasmids remain `cryptic` in that functional genes have not been isolated from them, hence leaving their functional significance speculative (Hishinuma et al., J. Gen. Microbiol. 104:193-199 (1978); Eberhard et al., Plasmid6:1-6 (1981); Vasquez et al., FEBS Lett. 158:339-342 (1983)). Common genes found in other plasmids include those encoding plasmid replication and cellular maintenance, antibiotic resistance, bacteriocin production, sex determination, and other cellular functions (Kornberg and Baker, `DNA Replication`, 2.sup.nd ed. (1991)). Of particular interest to molecular biologists are plasmids that harbor restriction-modification (R-M) systems.
R-M systems occur naturally in most bacteria, including thermophiles (Hjorleifsdottir et al., Biotech. Tech. 10:13-18 (1996)). The common type II R-M system consists of two genes encoding a restriction endonuclease and its cognate modification methylase (Roberts and Halford, in `Nucleases`, .sub.2 nd ed., Linn et al., ed.'s, p. 35-88 (1993)). When purified from other bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments for molecular cloning and gene characterization. Thermophilic restriction endonucleases tend to retain their thermophilic character, acting with maximum efficiency at the elevated temperatures of their host strain (Hjorleifsottir et al., Biotech. Tech. 10:13-18 (1996)). Thermophilic enzymes are also more stable than lower-temperature counterparts, being more resistant to both thermal and chemically induced denaturation (Kristjansson, Trends. Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992); Cowan, Biochem. Soc. Symp. 58:149-169 (1992)). They are therefore invaluable in methodology, such as PCR, in which high temperatures cannot be avoided.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within or to one side of the recognition sequence. Over two hundred and thirty two restriction endonucleases with unique specificity have been identified in bacterial species to date. Only about thirty of the over two thousand eight hundred known restriction endonucleases are known to be plasmid-borne (Roberts and Macelis, Nucl. Acids Res. 25:248-262, (1997)).
Restriction endonucleases are typically named according to the bacteria from which they are derived (Smith and Nathans, J. Mol. Biol. 81:419-423 (1973)). Thus, the Thermus species YS45 possesses one known endonuclease activity called Tsp45I (Raven et al., Nucl. Acids Res. 21:4397 (1993)). This enzyme recognizes two unique double-stranded DNA sequences: 5'-GTGAC-3' and 5'-GTCAG-3' (which can be conveniently written as 5'-GTSAC-3'). It cleaves the DNA before the first G in this site (along both strands) leaving four nucleotides as single stranded 5' overhangs at each end of the cleaved DNA. The enzyme has maximal activity at about 65.degree., a temperature at which YS45 grows well.
It is commonly accepted that restriction endonucleases evolved to play a protective role in the welfare of the bacterial cell (Wilson and Murray, Annu. Rev. Genet. 25:585-627 (1991)). They impart bacteria with resistance to infection by foreign viral or plasmid DNA, which might otherwise destroy or parasitize them. Invading foreign DNA is cleaved at recognition sites by the bacterial endonuclease, disabling many infecting genes and/or rendering the foreign DNA susceptible to further degradation by non-specific nucleases.
The other component of type II bacterial R-M systems is the modification methylase (Roberts and Halford, in `Nucleases`, .sub.2 nd ed., Linn et al., ed.'s, p. 35-88 (1993)). Modification methylases provide the means by which bacteria are able to protect and distinguish their own DNA from foreign DNA. They recognize and bind to the same recognition sequence as their corresponding restriction endonuclease. For example, the methylase recognizing 5'-GTSAC-3' is known as M. Tsp45I. Modification methylases do not cleave DNA, but rather chemically modify one particular nucleotide within the recognition sequence by the addition of a methyl group. Following methylation, this sequence is no longer cleaved by the corresponding endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified and therefore identifiably foreign DNA that is sensitive to restriction endonuclease recognition and cleavage.
It is often particularly difficult to cultivate thermophilic bacteria within the laboratory. They require high temperatures and often-unknown environmental conditions for acceptable growth (Kristjansson and Stetter, in `Thermophilic Bacteria`, Kristjansson, ed., p. 1-18 (1992)). However, with the advent of genetic engineering, it is now possible to clone genes from thermophiles into more easily cultivatable laboratory organisms, such as E. coli (Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). The expression of such genes can be finely controlled within E. coli.
A number of methods for isolating R-M systems from diverse bacteria have been devised. The earliest cloning efforts relied upon bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet. 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); Pstl: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Cells that carry cloned R-M genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method is of limited value, as many cloned R-M genes do not manifest sufficient phage resistance to confer selective survival. The likelihood of cloning a Thermus R-M system by this method is further reduced, as only one Thermus phage (fYS40) has been described (Sakaki and Oshima, J. Virol. 15:1449-1453, (1975)).
R-M systems have also been cloned by selection for an active methylase (`methylase-selection` (Kiss et al., Nucl. Acids Res. 13: 6403-6421, (1985)), or endonuclease (`endo-blue method`, (Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). These methodologies rely upon the expression of said genes in E. coli by their introduced promoters. Thermus promoters can significantly diverge from those of E. coli (Maseda and Hoshino, FEMS Microbiol. Lett. 128:127-134, (1995)), and may not function at all (Wayne and Xu, Gene (in press), (1997)). It is therefore difficult to predict whether such methodology can be used to clone a Thermus R-M system.
A few plasmid-borne R-M systems have been characterized in diverse bacterial species prior to transfer to E. coli (EcoRV, Bougueleret et al., Nucl. Acids Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Nat. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985)). However, no eubacterial, and only one archaeon (MthTI, Nolling and DEVOS, J. Bacteriol. 17:5719-5726 (1992)) thermophilic plasmid-borne R-M system has been previously expressed in E. coli. The cloning of thermostable proteins, such as restriction endonucleases, has been hampered by the lack of molecular methodologies within thermophiles. It is often simpler to clone said genes into E. coli prior to functional characterization (Kirino et al., Eur. J. Biochem. 220:275-281 (1994); Moriyama et al., J. Biochem. 117:408-413 (1995); Numata et al., Prot. Eng. 8:39-43 (1995)).
The purification of recombinant proteins from E. coli has also been better established than that from thermophiles.
Therefore, the production of recombinant proteins is often simpler and produces larger yields than those obtained through conventional purification from the original thermophile (Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992); Ishida and Oshima, J. Bacteriol. 176:2767-2770 (1994)). There is commercial incentive to produce thermostable endonucleases which are usually more stable to heat and denaturing conditions then mesophilic (grow between 20.degree. and 50.degree. C.) counterparts (Wiegel and Ljungdahl, CRC Crit. Rev. Biotech. 3:39-108); Kristjansson, Trends Biotech. 7:349-353 (1989); Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992)). These thermostable enzymes can also be used in a variety of assays, such as PCR, in which high temperatures cannot be 'avoided. The plasmids of thermophiles are therefore an appropriate source for finding thermophilic R-M systems.